Coupling the Antimalarial Cell Penetrating Peptide TP10 to Classical Antimalarial Drugs Primaquine and Chloroquine Produces Strongly Hemolytic Conjugates.

Recently, we disclosed primaquine cell penetrating peptide conjugates that were more potent than parent primaquine against liver stage Plasmodium parasites and non-toxic to hepatocytes. The same strategy was now applied to the blood-stage antimalarial chloroquine, using a wide set of peptides, including TP10, a cell penetrating peptide with intrinsic antiplasmodial activity. Chloroquine-TP10 conjugates displaying higher antiplasmodial activity than the parent TP10 peptide were identified, at the cost of an increased hemolytic activity, which was further confirmed for their primaquine analogues. Fluorescence microscopy and flow cytometry suggest that these drug-peptide conjugates strongly bind, and likely destroy, erythrocyte membranes. Taken together, the results herein reported put forward that coupling antimalarial aminoquinolines to cell penetrating peptides delivers hemolytic conjugates. Hence, despite their widely reported advantages as carriers for many different types of cargo, from small drugs to biomacromolecules, cell penetrating peptides seem unsuitable for safe intracellular delivery of antimalarial aminoquinolines due to hemolysis issues. This highlights the relevance of paying attention to hemolytic effects of cell penetrating peptide-drug conjugates.

Antimalarial Benzoxaboroles Target Plasmodium falciparum Leucyl-tRNA Synthetase.

There is a need for new antimalarials, ideally with novel mechanisms of action. Benzoxaboroles have been shown to be active against bacteria, fungi, and trypanosomes. Therefore, we investigated the antimalarial activity and mechanism of action of 3-aminomethyl benzoxaboroles against Plasmodium falciparum Two 3-aminomethyl compounds, AN6426 and AN8432, demonstrated good potency against cultured multidrug-resistant (W2 strain) P. falciparum (50% inhibitory concentration [IC50] of 310 nM and 490 nM, respectively) and efficacy against murine Plasmodium berghei infection when administered orally once daily for 4 days (90% effective dose [ED90], 7.4 and 16.2 mg/kg of body weight, respectively). To characterize mechanisms of action, we selected parasites with decreased drug sensitivity by culturing with stepwise increases in concentration of AN6426. Resistant clones were characterized by whole-genome sequencing. Three generations of resistant parasites had polymorphisms in the predicted editing domain of the gene encoding a P. falciparum leucyl-tRNA synthetase (LeuRS; PF3D7_0622800) and in another gene (PF3D7_1218100), which encodes a protein of unknown function. Solution of the structure of the P. falciparum LeuRS editing domain suggested key roles for mutated residues in LeuRS editing. Short incubations with AN6426 and AN8432, unlike artemisinin, caused dose-dependent inhibition of [(14)C]leucine incorporation by cultured wild-type, but not resistant, parasites. The growth of resistant, but not wild-type, parasites was impaired in the presence of the unnatural amino acid norvaline, consistent with a loss of LeuRS editing activity in resistant parasites. In summary, the benzoxaboroles AN6426 and AN8432 offer effective antimalarial activity and act, at least in part, against a novel target, the editing domain of P. falciparum LeuRS.

Cryptosporidium and Toxoplasma Parasites Are Inhibited by a Benzoxaborole Targeting Leucyl-tRNA Synthetase.

The apicomplexan parasites Cryptosporidium and Toxoplasma are serious threats to human health. Cryptosporidiosis is a severe diarrheal disease in malnourished children and immunocompromised individuals, with the only FDA-approved drug treatment currently being nitazoxanide. The existing therapies for toxoplasmosis, an important pathology in immunocompromised individuals and pregnant women, also have serious limitations. With the aim of developing alternative therapeutic options to address these health problems, we tested a number of benzoxaboroles, boron-containing compounds shown to be active against various infectious agents, for inhibition of the growth of Cryptosporidium parasites in mammalian cells. A 3-aminomethyl benzoxaborole, AN6426, with activity in the micromolar range and with activity comparable to that of nitazoxanide, was identified and further characterized using biophysical measurements of affinity and crystal structures of complexes with the editing domain of Cryptosporidium leucyl-tRNA synthetase (LeuRS). The same compound was shown to be active against Toxoplasma parasites, with the activity being enhanced in the presence of norvaline, an amino acid that can be mischarged by LeuRS. Our observations are consistent with AN6426 inhibiting protein synthesis in both Cryptosporidium and Toxoplasma by forming a covalent adduct with tRNA(Leu) in the LeuRS editing active site and suggest that further exploitation of the benzoxaborole scaffold is a valid strategy to develop novel, much needed antiparasitic agents.

Novel squaramides with in vitro liver stage antiplasmodial activity.

A structure-activity relationship study was performed with ten 8-aminoquinoline-squaramides compounds active against liver stage malaria parasites, using human hepatoma cells (Huh7) infected by Plasmodium berghei parasites. In addition, their blood-schizontocidal activity was assessed against chloroquine-resistant W2 strain Plasmodium falciparum. Compound 3 was 7.3-fold more potent than the positive control primaquine against liver-stage parasites, illustrating the importance of the squarate moiety to activity.

Chemical genetics of Plasmodium falciparum.

Malaria caused by Plasmodium falciparum is a disease that is responsible for 880,000 deaths per year worldwide. Vaccine development has proved difficult and resistance has emerged for most antimalarial drugs. To discover new antimalarial chemotypes, we have used a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library-many of which showed potent in vitro activity against drug-resistant P. falciparum strains-and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in several organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Our findings provide the scientific community with new starting points for malaria drug discovery.

Impact of antimalarial treatment and chemoprevention on the drug sensitivity of malaria parasites isolated from ugandan children.

Changing treatment practices may be selecting for changes in the drug sensitivity of malaria parasites. We characterized ex vivo drug sensitivity and parasite polymorphisms associated with sensitivity in 459 Plasmodium falciparum samples obtained from subjects enrolled in two clinical trials in Tororo, Uganda, from 2010 to 2013. Sensitivities to chloroquine and monodesethylamodiaquine varied widely; sensitivities to quinine, dihydroartemisinin, lumefantrine, and piperaquine were generally good. Associations between ex vivo drug sensitivity and parasite polymorphisms included decreased chloroquine and monodesethylamodiaquine sensitivity and increased lumefantrine and piperaquine sensitivity with pfcrt 76T, as well as increased lumefantrine sensitivity with pfmdr1 86Y, Y184, and 1246Y. Over time, ex vivo sensitivity decreased for lumefantrine and piperaquine and increased for chloroquine, the prevalences of pfcrt K76 and pfmdr1 N86 and D1246 increased, and the prevalences of pfdhfr and pfdhps polymorphisms associated with antifolate resistance were unchanged. In recurrent infections, recent prior treatment with artemether-lumefantrine was associated with decreased ex vivo lumefantrine sensitivity and increased prevalence of pfcrt K76 and pfmdr1 N86, 184F, and D1246. In children assigned chemoprevention with monthly dihydroartemisinin-piperaquine with documented circulating piperaquine, breakthrough infections had increased the prevalence of pfmdr1 86Y and 1246Y compared to untreated controls. The noted impacts of therapy and chemoprevention on parasite polymorphisms remained significant in multivariate analysis correcting for calendar time. Overall, changes in parasite sensitivity were consistent with altered selective pressures due to changing treatment practices in Uganda. These changes may threaten the antimalarial treatment and preventive efficacies of artemether-lumefantrine and dihydroartemisinin-piperaquine, respectively.

Tetrafluorophenoxymethyl ketone cruzain inhibitors with improved pharmacokinetic properties as therapeutic leads for Chagas' disease.

Inhibition of the cysteine protease cruzain from Trypanosoma cruzi has been studied pre-clinically as a new chemotherapeutic approach to treat Chagas' disease. Efficacious effects of vinylsulfone-based cruzain inhibitors in animal models support this therapeutic hypothesis. More recently, substrate-activity screening was used to identify nonpeptidic tetrafluorophenoxymethyl ketone inhibitors of cruzain that showed promising efficacy in animal models. Herein we report efforts to further optimize the in vitro potency and in vivo pharmacokinetic properties of this new class of cruzain inhibitors. Through modifications of the P1, P2 and/or P3 positions, new analogs have been identified with reduced lipophilicity, enhanced potency, and improved oral exposure and bioavailability.

N10,N11-di-alkylamine indolo[3,2-b]quinolines as hemozoin inhibitors: design, synthesis and antiplasmodial activity.

We recently reported that potent N10,O11-bis-alkylamine indolo[3,2-b]quinoline antimalarials act as hemozoin (Hz) growth inhibitors. To improve access and binding to the target we have now designed novel N10,N11-di-alkylamine bioisosteres. 3-Chloro derivatives (10a-f) showed selectivity for malaria parasite compared to human cells, high activity against Plasmodium falciparum chloroquine (CQ)-resistant strain W2 (IC50s between 20 and 158nM), good correlation with ß-hematin inhibition and improved vacuolar accumulation ratios, thus suggesting inhibition of Hz growth as one possible mechanism of action for these compounds. Moreover, our studies show that Hz is a valid target for the development of new antimalarials able to overcome CQ resistance.

Hsp90 inhibitors as new leads to target parasitic diarrheal diseases.

Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 µM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis.

1H-1,2,3-Triazole-tethered isatin-7-chloroquinoline and 3-hydroxy-indole-7-chloroquinoline conjugates: synthesis and antimalarial evaluation.

A series of 1H-1,2,3-triazole-tethered isatin-7-chloroquinoline and 3-hydroxy-indole-7-chloroquinoline conjugates have been synthesized and evaluated for their antimalarial activity against chloroquine-resistant W2 strain of Plasmodium falciparum. The most potent of the test compound with an optimum combination of 3-hydroxy-indole ring and a n-butyl linker displayed an IC50 value of 69 nM.

Synthesis, biological evaluation, hydration site thermodynamics, and chemical reactivity analysis of α-keto substituted peptidomimetics for the inhibition of Plasmodium falciparum.

A new series of peptidomimetic pseudo-prolyl-homophenylalanylketones were designed, synthesized and evaluated for inhibition of the Plasmodium falciparum cysteine proteases falcipain-2 (FP-2) and falcipain-3 (FP-3). In addition, the parasite killing activity of these compounds in human blood-cultured P. falciparum was examined. Of twenty-two (22) compounds synthesized, one peptidomimetic comprising a homophenylalanine-based α-hydroxyketone linked Cbz-protected hydroxyproline (39) showed the most potency (IC50 80 nM against FP-2 and 60 nM against FP-3). In silico analysis of these peptidomimetic analogs offered important protein-ligand structural insights including the role, by WaterMap, of water molecules in the active sites of these protease isoforms. The pseudo-dipeptide 39 and related compounds may serve as a promising direction forward in the design of competitive inhibitors of falcipains for the effective treatment of malaria.

A reprofiled drug, auranofin, is effective against metronidazole-resistant Giardia lamblia.

Giardiasis is one of the most common causes of diarrheal disease worldwide. Treatment is primarily with 5-nitro antimicrobials, particularly metronidazole. Resistance to metronidazole has been described, and treatment failures can occur in up to 20% of cases, making development of alternative antigiardials an important goal. To this end, we have screened a chemical library of 746 approved human drugs and 164 additional bioactive compounds for activity against Giardia lamblia. We identified 56 compounds that caused significant inhibition of G. lamblia growth and attachment. Of these, 15 were previously reported to have antigiardial activity, 20 were bioactive but not approved for human use, and 21 were drugs approved for human use for other indications. One notable compound of the last group was the antirheumatic drug auranofin. Further testing revealed that auranofin was active in the low (4 to 6)-micromolar range against a range of divergent G. lamblia isolates representing both human-pathogenic assemblages A and B. Most importantly, auranofin was active against multiple metronidazole-resistant strains. Mechanistically, auranofin blocked the activity of giardial thioredoxin oxidoreductase, a critical enzyme involved in maintaining normal protein function and combating oxidative damage, suggesting that this inhibition contributes to the antigiardial activity. Furthermore, auranofin was efficacious in vivo, as it eradicated infection with different G. lamblia isolates in different rodent models. These results indicate that the approved human drug auranofin could be developed as a novel agent in the armamentarium of antigiardial drugs, particularly against metronidazole-resistant strains.

In vitro and in vivo antimalarial efficacies of optimized tetracyclines.

With increasing resistance to existing antimalarials, there is an urgent need to discover new drugs at affordable prices for countries in which malaria is endemic. One approach to the development of new antimalarial drugs is to improve upon existing antimalarial agents, such as the tetracyclines. Tetracyclines exhibit potent, albeit relatively slow, action against malaria parasites, and doxycycline is used for both treatment (with other agents) and prevention of malaria. We synthesized 18 novel 7-position modified tetracycline derivatives and screened them for activity against cultured malaria parasites. Compounds with potent in vitro activity and other favorable drug properties were further tested in a rodent malaria model. Ten compounds inhibited the development of cultured Plasmodium falciparum with a 50% inhibitory concentration (IC50) after 96 h of incubation of <30 nM, demonstrating activity markedly superior to that of doxycycline (IC50 at 96 h of 320 nM). Most compounds showed little mammalian cell cytotoxicity and no evidence of in vitro phototoxicity. In a murine Plasmodium berghei model, 13 compounds demonstrated improved activity relative to that of doxycycline. In summary, 7-position modified tetracyclines offer improved activity against malaria parasites compared to doxycycline. Optimized compounds may allow lower doses for treatment and chemoprophylaxis. If safety margins are adequate, dosing in children, the group at greatest risk for malaria in countries in which it is endemic, may be feasible.

In vitro efficiency of 9-(N-cinnamoylbutyl)aminoacridines against blood- and liver-stage malaria parasites.

Novel 9-aminoacridine derivatives were synthesized by linking the heteroaromatic core to different cinnamic acids through an aminobutyl chain. The test compounds demonstrated mid-nanomolar in vitro activity against erythrocytic stages of the chloroquine-resistant W2 strain of the human malaria parasite Plasmodium falciparum. Two of the most active derivatives also showed in vitro activity against liver-stage Plasmodium berghei, with activity greater than that of the reference liver-stage antimalarial primaquine. The compounds were not toxic to human hepatoma cells at concentrations up to 5 µM. Hence, 9-(N-cinnamoylbutyl)aminoacridines are a new class of leads for prevention and treatment of malaria.

Novel anti-plasmodial hits identified by virtual screening of the ZINC database.

Increased resistance of Plasmodium falciparum to most available drugs challenges the control of malaria. Studies with protease inhibitors have suggested important roles for the falcipain family of cysteine proteases. These enzymes act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. In order to find potential new antimalarial drugs, we screened in silico the ZINC database using two different protocols involving structure- and ligand-based methodologies. Our search identified 19 novel low micromolar inhibitors of cultured chloroquine resistant P. falciparum. The most active compound presented an IC50 value of 0.5 µM against cultured parasites and it also inhibited the cysteine protease falcipain-2 (IC50 = 25.5 µM). These results identify novel classes of antimalarials that are structurally different from those currently in use and which can be further derivatized to deliver leads suitable for optimisation.

Tetrazole-based deoxyamodiaquines: synthesis, ADME/PK profiling and pharmacological evaluation as potential antimalarial agents.

A series of new deoxyamodiaquine-based compounds was synthesized via the modified TMSN3-Ugi multi-component reaction and evaluated in vitro for antiplasmodial activity. The most potent compounds, 6b, 6c and 6j, showed IC50 values in the range of 6-77nM against chloroquine-resistant K1- and W2-strains of Plasmodium falciparum. In vitro ADME characterization of frontrunner compounds 6b and 6c indicates that these two compounds are rapidly metabolized and have a high clearance rate in human and rat liver microsomes. This result correlated well with an in vivo pharmacokinetics study, which showed low bioavailability of 6c in rats. Tentative metabolite identification was determined by LC-MS and suggested metabolic lability of groups attached to the tertiary nitrogen. Preliminary studies on 6b and 6c suggested strong inhibitory activity against the major CYP450 enzymes. In silico docking studies were used to rationalize strong inhibition of CYP3A4 by 6c. Full characterization and biological evaluation of the metabolites is currently underway in our laboratories.

Chemical-biological characterization of a cruzain inhibitor reveals a second target and a mammalian off-target.

Inhibition of the Trypanosoma cruzi cysteine protease cruzain has been proposed as a therapeutic approach for the treatment of Chagas' disease. Among the best-studied cruzain inhibitors to date is the vinylsulfone K777 (1), which has proven effective in animal models of Chagas' disease. Recent structure-activity studies aimed at addressing potential liabilities of 1 have now produced analogues such as N-[(2S)-1-[[(E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]amino]-3-(4-methylphenyl)-1-oxopropan-2-yl]pyridine-4-carboxamide (4), which is trypanocidal at ten-fold lower concentrations than for 1. We now find that the trypanocidal activity of 4 derives primarily from the inhibition of T. cruzi 14-α-demethylase (TcCYP51), a cytochrome P450 enzyme involved in the biosynthesis of ergosterol in the parasite. Compound 4 also inhibits mammalian CYP isoforms but is trypanocidal at concentrations below those required to significantly inhibit mammalian CYPs in vitro. A chemical-proteomics approach employing an activity-based probe derived from 1 was used to identify mammalian cathepsin B as a potentially important off-target of 1 and 4. Computational docking studies and the evaluation of truncated analogues of 4 reveal structural determinants for TcCYP51 binding, information that will be useful in further optimization of this new class of inhibitors.

Artefenomel Regioisomer RLA-3107 Is a Promising Lead for the Discovery of Next-Generation Endoperoxide Antimalarials.

Clinical development of the antimalarial artefenomel was recently halted due to formulation challenges stemming from the drug's lipophilicity and low aqueous solubility. The symmetry of organic molecules is known to influence crystal packing energies and by extension solubility and dissolution rates. Here we evaluate RLA-3107, a desymmetrized, regioisomeric form of artefenomel in vitro and in vivo, finding that the regioisomer retains potent antiplasmodial activity while offering improved human microsome stability and aqueous solubility as compared to artefenomel. We also report in vivo efficacy data for artefenomel and its regioisomer across 12 different dosing regimens.

Novel potent metallocenes against liver stage malaria.

Novel conjugates of the antimalarial drug primaquine (compound 1) with ferrocene, named primacenes, have been synthesized and screened for their activities against blood stage and liver stage malaria in vitro and host-vector transmission in vivo. Both transmission-blocking and blood-schizontocidal activities of the parent drug were conserved only in primacenes bearing a basic aliphatic amine group. Liver stage activity did not require this structural feature, and all metallocenes tested were comparable to or better than primaquine in this regard. Remarkably, the replacement of primaquine's aliphatic chain by hexylferrocene, as in compound 7, led to a ~45-fold-higher level activity against liver stage parasitemia than that of primaquine.

Compounds from Sorindeia juglandifolia (Anacardiaceae) exhibit potent anti-plasmodial activities in vitro and in vivo.

BACKGROUND: Discovering new lead compounds against malaria parasites is a crucial step to ensuring a sustainable global pipeline for effective anti-malarial drugs. As far as we know, no previous phytochemical or pharmacological investigations have been carried out on Sorindeia juglandifolia. This paper describes the results of an anti-malarial activity-driven investigation of the fruits of this Cameroonian plant. METHODS: Air-dried fruits were extracted by maceration using methanol. The extract was fractionated by flash chromatography followed by column chromatography over silica gel, eluting with gradients of hexane-ethyl acetate mixtures. Resulting fractions and compounds were tested in vitro against the Plasmodium falciparum chloroquine-resistant strain W2, against field isolates of P. falciparum, and against the P. falciparum recombinant cysteine protease falcipain-2. Promising fractions were assessed for acute toxicity after oral administration in mice. One of the promising isolated compounds was assessed in vivo against the rodent malaria parasite Plasmodium berghei. RESULTS: The main end-products of the activity-guided fractionation were 2,3,6-trihydroxy benzoic acid (1) and 2,3,6-trihydroxy methyl benzoate (2). Overall, nine fractions tested against P. falciparum W2 and falcipain-2 were active, with IC50 values of 2.3-11.6 µg/ml for W2, and 1.1-21.9 µg/ml for falcipain-2. Purified compounds (1) and (2) also showed inhibitory effects against P. falciparum W2 (IC50s 16.5 µM and 13.0 µM) and falcipain-2 (IC50s 35.4 and 6.1 µM). In studies of P. falciparum isolates from Cameroon, the plant fractions demonstrated IC50 values of 0.14-19.4 µg/ml and compounds (1) and (2) values of 6.3 and 36.1 µM. In vivo assessment of compound (1) showed activity against P. berghei strain B, with mean parasitaemia suppressive dose and curative dose of 44.9 mg/kg and 42.2 mg/kg, respectively. Active fractions were found to be safe in mice after oral administration of 7 g/kg body weight. CONCLUSIONS: Fractions of Sorindeia juglandifolia and two compounds isolated from these fractions were active against cultured malaria parasites, the P. falciparum protease falcipain-2, and in a rodent malaria model. These results suggest that further investigation of the anti-malarial activities of natural products from S. juglandifolia will be appropriate.